This invention relates to primers, probes, assay kits and methods for identifying and discriminating between interferon (IFN) subtypes.
Interferons are secreted cellular proteins that are comprised of type I, type II and type III families based upon sequence similarities and receptor usage. IFN-γ, known also as immune interferon, is the only Type II interferon whereas the Type I human interferons consist of several classes: IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ and IFN-τ. IFN-τ is only found in ungulates; there is no human IFN-τ. There is only one human IFN-β and one human IFN-ω, but a family of multiple IFN-α species exists, namely, thirteen highly homologous genes, two of which have an identical coding sequence. IFNs are up-regulated in response to varied native and pathologic stimuli. Pathogens such as viruses are recognized by components of the innate immune system including toll-like receptors (TLR) and retinoic acid-inducible gene-1-like (RIG-1) receptors. The rapid recognition of pathogen stimuli coordinated with the appropriate cytokine response are crucial for the control of many pathogenic organisms. To illustrate, rather than a single interferon responding to a specific infectious agent, the host interferon response comprises coordinated expression of many different interferon genes and proteins designed a) to limit pathogen replication in infected cells and b) to coordinate with the adaptive immune system to limit dissemination. This “interferon expression signature” varies over time, coordinating and responding to the expression of thousands of genes.
Importantly, it has been clearly established in the literature that each individual interferon protein possesses unique biological activities. Moreover, limited qualitative studies have shown that the expression patterns of the interferon alpha subtypes vary with stimuli and time. Consequently, assays are warranted that can detect and differentiate each of the individual interferons.
The IFNs exhibit anti-viral, immunoregulatory, and antiproliferative activity, and the clinical potential of interferons has been recognized. However, little is known about unique roles for any of the individual IFN-alpha subtypes because there is no sensitive and specific system to measure their individual expression.
Thus, one of the current research dilemmas within the interferon world is the role of the various IFN types and subtypes in various diseases. Currently, tools are not available to address these questions, although several pharmaceutical companies are investigating the modulation of individual IFN subtypes as potential therapies for a variety of human diseases, including systemic lupus erythematosis, multiple sclerosis, various cancers, hepatitis C, human immunodeficiency virus (HIV) and others. The availability of tools which address both the individual and combined expression profiles of interferons and/or other cytokines will greatly benefit not only current research, but will enable other areas of disease research where interferons have not even been implicated due to a lack of suitable tools.
The invention described herein provides nucleic acid amplification techniques for differentiating between highly related nucleic acid molecules. In current embodiments, polymerase chain reaction (PCR) and novel probe/primer pairs are used to differentiate between IFN subtypes. The invention described herein will be useful for elucidating fundamental roles of various closely related interferons and subtypes and, in the process, greatly advance what is known about immunological processes particularly in the context of the subtle genetic differences between humans. The invention is expected to be useful in relation to other biological molecules for which different, highly related subtypes occur in the body. The instant invention may be useful, for example, for identifying subtypes of IFN as a surrogate marker for vaccine efficacy in clinical trials, to monitor clinical progression or remission of chronic infections, autoimmune diseases or cancer, or to determine responses to therapy for those and other diseases. Moreover, the instant invention may be useful, inter alia, in molecular diagnostic methods and in enabling skilled professionals to individually design and tailor therapeutic treatment regimens for the individual patient and the particular medical condition presented. The ability to deliver personalized medicine may be especially important in treating individuals who are refractory to the standard regiment of care. Thus, there is a need for a rapid comprehensive test for determining the presence of IFN-subtypes.